The invention relates to a method for applying a film of sample to a sample carrier for subsequent spectroscopic analysis.
In the context of spectroscopic sample analysis, in particular in the context of vibrational spectroscopy, such as for example IR, NIR, Raman spectroscopy, a method as mentioned above is used to prepare samples for the subsequent spectroscopic analysis.
The samples which are to be applied to the sample carrier may, for example, be biological liquids, such as serum, urine, suspended cells, cell culture media, etc. or analytes dissolved in water, which are subsequently to be analyzed and in particular quantified by IR spectroscopy.
On account of the high inherent absorption of some solvents in the infrared (IR) spectral region, the quantity of sample applied to the sample carrier in the liquid state is dried to form a film of sample prior to the spectroscopic measurement. This ideally leads to the formation of homogeneous films of sample, i.e. films of sample which have a uniform layer thickness, so that the Lambert-Beer law applies. However, this method of applying a film of sample by drying a quantity of sample applied in liquid form is unsuitable for many solvents, in particular water, since the film of sample which is formed by drying does not have a uniform layer thickness over the area of the sample position, but rather has a greater layer thickness toward the edge than in the center. This phenomenon is explained below with reference to FIGS. 3 and 4.
FIG. 3 illustrates a sample carrier 1 which has a sample position A to which a quantity of sample 2 in the liquid state has been applied. The quantity of sample typically has a volume of 1-100 μl, while the sample position A has a diameter of approximately 1-20 mm. The sample position A is the region of the sample carrier which, in the case, for example, of IR spectroscopy, is illuminated by the light beam during the subsequent spectroscopic analysis.
Now, if the quantity of sample, as illustrated in FIG. 3, is applied to the sample position A of the sample carrier 1 distributed uniformly in the form of a drop, the quantity of liquid sample, irrespective of whether it is applied manually using a pipette or with the aid of an automated pipetting device, in section forms approximately the shape of a half-oval on the sample position. The surface tension of the quantity of liquid sample in this case determines the precise shape of the half-oval.
After the quantity of sample 2 has been dried, what remains is a film of sample 3 which is significantly thicker at the edges than in the center, as illustrated in FIG. 4. Accordingly, the film of sample 3 has a crater-like appearance. This cratering effect is particularly pronounced if the solvent used for the sample is water. The layer thickness of the film of sample 3 formed is therefore not homogeneous across the sample position A.
It has emerged that when the same sample and the same quantity of sample are repeatedly discharged onto a plurality of sample positions, the films of sample which are formed after drying also often adopt different forms. This means that the known method for applying a film of sample to a sample carrier not only leads to an inhomogeneous layer thickness in each individual film of sample, but also leads to very different spectra, which may differ significantly in particular in terms of their signal intensity, being obtained during a spectroscopic analysis of a plurality of films of sample which have been produced from the same sample batch. Therefore, the conventional method has the drawback that the films of sample cannot be produced in an unambiguously reproducible way.